Summary: The aims of our experimental marrow stem cell transplantation (SCT) program are firstly to improve the outcome after allogeneic SCT by optimizing the stem cell and lymphocyte doses of the transplant, and secondly to exploit the antitumor effect of donor immune cells to treat hematological and non-hematological malignant diseases by adoptive immunotherapy. Transplant-related mortality in current transplant protocols is around 15% which represents a significant advance in developments which have improved the safety of the transplant. The current challenge is to improve the ability of the transplant to prevent relapse of the malignancy. This is being addressed in several clinical protocols and in laboratory research: Study 1 involves transplants for patients with hematological malignancy receiving stem cells from a matched sibling donor. A total of 37 patients have been transplanted on this protocol, five died from relapse five from transplant-related causes and 27 survive. Best results are achieved in patients with standard risk or intermediate risk for relapse. This group of 25 patients have an approximate survival of 80% compared with less than 30% for high-risk patients. Study 2 involves matched sibling transplants for older patients with hematological malignancy. A new technique (selective T cell depletion - SD) is being evaluated for its ability to reconstitute immunity without causing graft-versus-host disease (GVHD) after transplant. Twenty patients have been transplanted. One rejected the transplant but only two patients developed significant acute GVHD following transplant indicating a proof of principle that SD can be effective. The relapse rate is around 50% or this group of patients who are at high risk for disease progression and recurrence. Study 3 explores a transplant regimen using mismatched donors to establish engraftment without causing GVHD. Three patients have been transplanted. All patients engrafted and GVHD has been well controlled, however all patients died from infectious complications or disease relapse These results suggest that improvements in future studies might be achieved with SD transplants for mismatched individuals since immune recovery in study 2 has been excellent and GVHD is reduced by SD. Our laboratory research has several linked objectives: 1) the detection and characterization of lymphocytes with cytotoxicity against leukemia cells. 2) the discovery of new leukemia antigens capable of initiating a graft-versus-leukemia (GVL) reaction and also antigens which cause GVHD. 3) Development of techniques to select and expand leukemia-reacting and viral-reactive T cells for adoptive immunotherapy of patients with malignant diseases and to treat viruses which reactivate after transplant. 4) The direct monitoring in vivo of specific T cell responses to leukemia, tumor and viral antigens to characterize the occurrence of such antigen-specific T cells in healthy individuals, patients with leukemia and to follow immune responses after transplant and vaccine treatments. Advances in the last year have been in the area of translational research: (1) We have explored two new approaches to SD - firstly the use of a techniques called photodepletion which is based on the inability of activated T cells to extrude a rhodamine-based dye, which releases electrons when exposed to light. Activated T cells causing GVHD can thus be selectively removed by light exposure. Secondly we have studied the use of a magnetic bead column to remove activated T cells expressing CD25. The aim of this research is to create a readily available, highly efficient SD technique to apply to all future transplants. (2) We have developed a means to transfect antigen presenting cells with plasmids containing DNA sequences of cytomegalovirus (CMV) and leukemia antigens WT1 and PR3. We have used expanded T cells as a readily available form of aantigen-presenting cell. We have shown that autologous expanded T cells make excellent antigen-presenting cells. The aim is to continue translational research to generate antigen-specific T cells as adoptive immunotherapy after transplant to treat or prevent CMV disease and prevent leukemic relapse. (3) Vaccines. We have generated clinical grade vaccines of leukemia antigens WT1 and PR3 and are currently seeking regulatory approval to start a vaccine trial in patients with myeloid leukemia and related malignancies. (4) We are testing a CMV vaccine in donors of transplant patients to study whether boosted antiviral immunity in the donor can be transmitted to the recipient. If it is possible to expand the recall ressponse to CMV in this way it suggests that immune responses to leukemia specific antigens might be similarly be boosted. Important discoveries in the last year have been (1) The identification of a natural killer cell anti- leukemia effect in lymphocytes from patients with chronic myelogenous leukemia suggesting new mechanisms whereby natural-killer cells can regulate leukemic proliferation. (2) We have studied immune recovery after allogeneic stem cell transplantation. We found that regulatory T cells only slowly recover after transplant. This lack of regulation of immune responsivelenss may be the basis for the clonal T cell expansions observed after transplant and the occurrence of acute graft-versus-host disease. The early post-transplant immune environment may be conducive to vaccine-induced T cell expansion and supports strategies to give vaccination to boost antileukemic responses after transplant using PR3 and WT1 vaccines.(3) Ongoing studies in chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib has shown great promise in the treatment of CML However it is now clear that the agent does not eradicate the leukemia. It is possible that the most primitive progenitor cells for CML are not susceptible to the drug. However the ability to cure the disease by stem cell transplantation suggests that the Graft-versus-leukemia effect is capble of eradiating the most primitive CML stem cells. We are now using flow cytometric sorting techniques to dissect out the stem cell compartment of CML to identify whethrt ther is a progenitor subpopulation which is susceptible to immune control but not to imatinib. These findings have implicatins for the use of vacciens to boost immune responses to CML and thereby eradicate residual disease once the imatinib response is maximized.